Vitellogenin is a female-specific glucolipoprotein yolk precursor produced by all oviparous, egg-laying, animals. It is expressed under hormonal control, normally in the presence of estrogen or estrogen-like hormones, and is generally synthesized in the liver directly before yolk deposition (Amadan et. al, 2002). Although this protein is only naturally produced by females, both sexes have the vitellogenin gene; therefore, males are able to produce vitellogenin under special conditions (Tyler et. al, 1996). Because of the lack in estrogen in males, they only produce vitellogenin with outside simulation such as: injections of estradiol or ingestion of estrogen-like substances: DDT, DES and many more (Thomas Di Giulio and Hinton, 2008).
To illustrate the induction of the central dogma through estradiol, we used a model system, the production of vitellogenin in male Fundulus grandis.
METHODS
Animals Used
Approximately 15 fish, male and female, of the species Fundulus grandis were collected from Cocachie, LA and bought to Nicholls’ Climate Control lab. Throughout the duration of our experiment, the fish were kept in 15 ppt fresh water, checked-on, and cared for daily.
Experimental Procedure
Our experiment took place over a 3 week period. During week 1, the fish were separated according to their sex, then, the males were separated according to whether they were going to be our control or our treatment injected subjects. We had 6 fish for each group. Each of the separate groups were sedated for 2 minutes in 0.1g/l of MS-222 and weighed in grams. The weight was recorded. Next, we injected the fish with 0.01 mg/g body weight with either peanut oil (control) or with estradiol (E2) peanut oil (treatment). The injected fish were placed back into their specific tanks and left alone for a two week period. On week 3 the fish were once again sedated in 0.1g/L of MS-222 and weighed. After, we removed the tail at the caudal peduncle and transferred the blood collecting into 1.5 mL capillary tubes. An estimated 2 ½ tubes of blood were collected per fish. The tubes were blown into 50 mL tubes and centrifuged at 1 RCF for 3 minutes to collect the supernatant (serum) in order to assay for proteins in the future. The fish were beheaded at the point behind the gills and their livers removed. The liver was separated into two pieces and one piece was placed in tube with RNA-STAT-60 (reserves RNA) and the other into a dry tube to be assayed for DNA. The tubes that contained RNA-STAT-60 underwent RT-PCR analysis and the tubes with the dry DNA underwent PCR analysis at temperatures 94°C, 45°C, and 72°C. Finally, the products of PCR and RT-PCR analysis, both underwent gel electrophoresis and the results were recorded.
RESULTS
We measured the mass of fish in both sample groups at week one and week three. The average mass for control fish in week one was 10.73g +/- 3.35 (S.D); and the average mass of this same group of fish at week three was 11.06g +/- 3.07 (S.D) as shown in Figure 1. A summary of our results is presented in Table 1.
The average mass for the treatment fish at week one was 10.38 +/- 3.77 (S.D); and the average mass at week three was 11.30 +/- 3.32 (S.D) as shown in Figure 2. A summary of our results are shown in Table 1.
We performed gel electrophoresis on our DNA products from PCR and our RNA products from RT-PCR. We found that in our PCR (3A), all of the lanes held products and in our RT-PCR (3B), only lanes 7-1o, estradiol- injected male fish, and lanes 14-15, female fish, held products. Figure 3 shows a picture representation of this information. A summary of our results are recorded in Table 1.
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Table 1. A summary of my results and interpretations.
DISCUSSION
The formation of the central dogma by Francis crick helped scientist understand one of the most influential processes in the animal body. It also allowed us to test whether the central dogma was inducible with estradiol by measuring the mass and levels of vitellogenin in control and treatment-injected male Fundulus grandis over a three week period.
During our experimental period, we found that the mass of our fish, male and female, increased. This means that they were in a safe environment and well-taken care of.
We found through our PCR results that both control males and treatment males had the gene necessary to code for vitellogenin, meaning that all nuclei had a genomic copy of the vitellogenin sequence in both sexes. Others have found that in most species of fish, especially those that are hermaphroditic, both sexes have the gene necessary for egg-development and egg-yolk development (Thomas Di Giulio and Hinton, 2008). This led us to believe that transcription, the first step in the central dogma, had occurred.
Our results from RT-PCR showed that the products of the treatment males matched the female product and that the control males had no products. Studies show that outside simulation, injection or ingestion can cause vitellogenin production in male species (Tyler et.al, 1996). This increase in the levels of cDNA in treatment-injected males led us to believe that RNA synthesis had occurred.
In conclusion, the levels of vitellogenin in mRNA in control males was considerably lower than the levels if mRNA in treatment males; the central dogma is inducible by estradiol.
References
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, 2008. How Cells Read the Genome: From DNA to Protein. In Molecular Biology of the Cell 5th Edition. Eds. Anderson M and Granum S. Garland Sciences, NY. pp 331-334.
Amdam G, Norberg K, Hagen A, Omholt S,2003. Social exploitation of vitellogenin. PNAS. 100: 1799-1802.
Doerfler W and Bohm P, 2006. The Central Dogma of Molecular Biology Revisited. In DNA methylation: basic mechanisms. Ed Rallison S. Springer Berlin Heidelberg, NY. pp 252-253.
Thomas Di Giulio R and Hinton D, 2008. Estrogenic Effects of Treated Sewage Effluent on Fish Steroids and Surfactants in English Rivers. In The Toxicology of Fishes. Eds. Thomas Di Giulio R and Hinton D. Taylor & Francis Group, FL. pp 971-973.
Tyler CR, Van der Eerden B, Jobling S, Panter G, Sumpter JP, 1996. Measurement of vitellogenin, a biomarker for exposure to oestrogenic chemicals in a wide variety of cyprind fish. J Comp Physiol B. 166:418-426.
